1. Kirk C. Lo MD, FRCSC Assistant Professor, Division of Urology University of Toronto Ontario, Canada In vitro sperm maturation Reality or science fiction? For fertility preservation in pre-pubertal male cancer patients

2. Clinical Case  8 year-old boy diagnosed with leukemia  Underwent 1 cycle of chemotherapy  Leukemia relapsed in both testes (biopsy proven)  Referred to Urology for bilateral orchiectomy – part of treatment protocol  Parents inquired about fertility preservation

3. Clinical Case  Can we offer fertility preservation for this boy?  What are the technical limitations?  What are the ethical/legal concerns?

4. Cancer in young adults and children  Estimated 1.4 million people were diagnosed with cancer in 2005  4% (55,000) are under the age of 35  Every year over 750 children (aged 0-15 years) and 10,000 adolescent and young adults are diagnosed with a malignancy (Canadian Cancer Society) http://www.seer.cancer.gov/statfact

5. Cancer and Fertility  A recent survey showed that 51% of men with cancer want children in the future  Only 20-50% of men recover spermatogenesis after cytotoxic therapy. Schover et al JCO, 2002 Carson et al, Hum Reprod, 1991 Lee et al, JCO, 2006 http://www.seer.cancer.gov/statfact

6. Fertility preservation should be discussed with cancer patients prior to their gonadotoxic therapies

7. Pre-pubertal Children  No active spermatogenesis  Can not bank mature sperm  Need alternative method of germ cell preservation

8. Pre-pubertal Testicular tissue harvest & cryopreservation

9. Testicular maturation Adult testis Pre-pubertal testis ?

10. In vivo maturation -spermatogonial stem cell transplantation - Xenograft model In vitro differentiation Experimental strategies

11. I. Germ Cell Transplantation Brinster RL, Science 2002

12. Injection of isolated spermatogonial cells  Extract spermatogonial stem cells from cryopreserved testes  Re-colonize the seminiferous tubules post cancer treatment

13. Colonization of Recipient Mouse by ROSA Donor Testis Cells Nagano, et al., Biol. Reprod. 60:1429, 1999 Two Months Two Weeks One DayOne Week Three Months One Month

14. Risk of Re-introduce Malignant Cells Jahnukainen et al, Cancer Res, 2001 Theoretical or real concern?  Intratesticular transplantation of testicular cells from Leukemic Rats cause transmission of Leukemia!

15. Bottom line:  We still do not have a method to isolate a pure population of spermatogonial stem cells  Germ cell transplantation in human is still experimental

16. II. Testis xenotransplantation Honaramooz et al Nature, 2002

17. Donor speciesSpermatogenesisCommentsReference Mouse (allograft)completeembryos offspring Honaramooz et al. 2002 Schlatt et al. 2003 HamstercompleteSchlatt et al. 2002 RabbitcompleteoffspringShinohara et al. 2002 PigcompleteembryosHonaramooz et al. 2002 Goat, Sheepcompletesperm recoveryHonaramooz et al. 2002 Dobrinski et al. 2003 CattlecompleteOatley et al. 2004 Rathi et al. 205 Catcompletesperm recoverySnedaker et al. 2004 HorsecompleteRathi et al. 2004 Rhesus monkeycompleteembryosHonaramooz et al. 2002 Human (adult) Human (fetal) Poor germ cell survival *germ cell survival Schlatt et al. 2006 Geens et al. 2006 Yu et al, 2006 Literature Update

18. II. Testis xenotransplantation  grafting human “adult” testicular tissue from testicular biopsy showed “limited” survival  superior survival of the germinal epithelium and spermatogonia when spermatogenesis was suppressed prior to grafting Schlatt et al, Hum Reprod 2006

19. II. Testis xenotransplantation  Wyns and colleagues (2008) harvested and cryopreserved from five pre-pubertal boys prior to chemotherapy.  Thawed testicular tissue transplanted into the scrotum of nude mice can survive for at least 6 months  Spermatogonial stem cell recovery rate was 3.7+5.5%.  Numerous pre-meiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells  But complete regeneration of normal spermatogenesis was not achieved Wyns et al, Human Reprod 2008

20. Xenotransplantation of Human fetal Testes  Human fetal testis (9-12 weeks gestation)  Graft harvested at 24 wks post graft Lo et al, ESHRE 2008

21. Results Treatment Group# of testes grafted# (%) of testes survived Castrated87 (87.5) Castrated + hCG77 (100) Non-castrated75 (71.4) Non-castrated + hCG86 (75) Lo et al, ESHRE 2008

22. Results: 24 weeks post graft Pre-graft 24 weeks post graft

23. II. Testis xenotransplantation  Demonstrated good survival of grafted human fetal and prepubertal testis  Maturation of seminiferous tubules in grafted human fetal testis  Promising technique  Germ cell “contamination” with xenotransplantation  Still experimental

24. III. In vitro maturation of germ cells  In vitro maturation of germ cells  From spermatogonia – spermatocyte – spermatid – mature spermatozoa  In vitro derivation of germ cells  From Embryonic Stem Cells - Haploid Germ Cells – sperm (?)

25. In vitro maturation of germ cells  Simple organ culture of testis tissue from variety of species can be maintained (Goldshmidt 1915, Champy 1920, Michailow 1937)  Progression arrested at meiosis  Improvement of the culture system with controlled temperature and microenvironment in the 1960 and 70’s by Anna and Emil Steinberger

26. In vitro maturation of germ cells  Co culturing testicular biopsy sample from azoospermic men with Vero cells, Cremades et al reported in vitro maturation of round spermatid to elongated spermatid and mature sperm (Cremades et al 1999)  True maturation vs. heterogeneous germ cell presence?

27. In vitro maturation of germ cells  Subsequent study showed a single human primary spermatocyte can undergo meiosis and differentiate into 4 round spermatids when co-cultured with Vero cells (Tanaka et al, 2003)  However, no mature sperm found in this study.

28. Copyright restrictions may apply. Stukenborg, J.-B. et al. Mol. Hum. Reprod. 2009 15:521-529; doi:10.1093/molehr/gap052 Experimental design of the soft agarculture system (SACS) and the methylcellulose culture system (MCS) 3-D Spermatogonia Culture System

29. Copyright restrictions may apply. Stukenborg, J.-B. et al. Mol. Hum. Reprod. 2009 15:521-529; doi:10.1093/molehr/gap052 Immunohistochemical and morphological analysis of cells from the soft agarculture system (SACS)

30. In vitro maturation of germ cells  Promising 3-D culture system – maturation of spermatogonia to sperm in the mouse model  Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

31. Take home message  Strategies to “mature” sperm in vivo and in vitro are still in the experimental stages  Animal models showed promising results – mature sperm (can be used for ICSI)  More studies are required

32. Future Challenges  Increase awareness and participation of oncologist in counseling cancer patient with respect to gamete preservation  Provide adequate facility and protocol to streamline the process  Multidisciplinary approach – oncologist, reproductive specialist, psychiatrist, social work, ethicist, legal department, etc

33. Thank You

35. III. In vitro derivation of germ cells  Embryonic stem (ES) cells can differentiate into germ cells in mice Geijsen et al, Nature 2004 Toyooka et al, PNAS 2003

36. Niels Geijsen,and George Q. Daley Nature 427, 148-154(8 January 2004) Derivation of embryonic germ cell & male gametes from mouse ES cells

37. Niels Geijsen,and George Q. Daley Nature 427, 148-154(8 January 2004) Blastocysts derived from Iintracytoplasmic injection of oocytes with EB-derived FE-J1 + haploid cells.

38. III. In vitro maturation of germ cells Navernia K et al, Dev Cell 2006

39. III. Derivation of germ cells from Human ES cells  Spontaneous differentiation of germ cells from human embryonic stem cells in vitro  Bone Morphogenetic protein (BMP) induce further germ cell differentiation from human ES cells Clark et al, Hum Mol Gene 2004 Kee et al, Stem Cell Dev 2006

40. Copyright restrictions may apply. Clark, A. T. et al. Hum. Mol. Genet. 2004 13:727-739; doi:10.1093/hmg/ddh088

41. Copyright restrictions may apply. Clark, A. T. et al. Hum. Mol. Genet. 2004 13:727-739; doi:10.1093/hmg/ddh088

42. K Kee et al. Nature 000, 1-4 (2009) doi:10.1038/nature08562 Overexpression of DAZL, DAZ and BOULE induces meiotic progression and haploid formation.

43. III. Derivation of germ cells from Human ES cells  Different approaches & culture systems have been reported to isolate and derive primordial germ cells and Sertoli cells from Human ES cells  Limited to the early stages  No mature sperm to date Tilgner et al, Stem cells 2008 Bucay N et al, stem cells 2009 Park TS et al, Stem cells 2009

44. III. In vitro maturation of germ cells  Nayernia and colleagues reported the derivation of human sperm from embryonic stem cells  Human ES cells were transfected with Stra8-EGFP  Cultured on a feeder layer, containing retinoic acid (RA).  Sorted GFP + cells express germ cell markers  Formed haploid sperm-like cells!!! Nayernia et al, Stem cells Dev, July 2009 epub

45. III. In vitro maturation of germ cells Nayernia et al, Stem cells Dev, July 2009 epub

46. Case 1  Consultation with the legal and ethical department  Informed consent from the patient and boy’s parents  Bilateral orchiectomy at SickKids hospital  Histology: fibrosis with presence of spermatogonial cells  Cryopreserved at RBU Mount Sinai Hospital  Continue chemotherapy…..